The development of Type 1 Diabetes (T1D) relies on complex interactions between genes imparting disease susceptibility and environmental factors. These interactions influence the cells of the immune system that mediate the autoimmune destruction of insulin-producing pancreatic ? cells. Despite the nearly 40 genes that have been identified to confer genetic risk for T1D, the functional impact of these specific gene variants remain poorly characterized. Our studies are designed to address this knowledge gap by providing an understanding for the mechanism by which CD226, a gene linked to risk for multiple autoimmune disorders, impacts immune regulation and may precipitate autoimmune disease in those at risk. CD226 is an immune costimulatory molecule that competes with an inhibitory receptor known as T-cell immunoreceptor with Ig and ITIM domain (TIGIT) for binding to CD155 or CD112 expressed on antigen presenting cells (APC). The CD226:TIGIT regulatory axis is analogous in many ways to the well characterized CD28 and CTLA-4 interaction that has resulted in potent immune modulating therapeutics for the treatment of cancer, transplantation, and autoimmunity. Our preliminary data suggest that CD226 expression on T cells is tightly linked to differentiated memory and effector T cell subsets that are widely thought to drive ? cel destruction. In addition, CD226 is expressed on a unique subset of regulatory T cells (Tregs) that exhibit epigenetic instability, reduced suppressive capacity, and produce cytokines. These data have led us to propose the hypothesis that disease-associated variants and inflammatory environmental triggers result in a functional imbalance in the CD226:TIGIT regulatory axis favoring immune activation by CD226 over regulation by TIGIT in T1D. We will test this hypothesis by conducting a series of Aims to i) determine the impact of modulating CD226 and TIGIT expression on T cell activation and regulation, ii) assessing the expression of CD226 and TIGIT throughout the natural history of T1D in the Non-Obese Diabetic mouse model and in humans with T1D, and iii) testing the therapeutic potential of targeting this axis to generate stable regulatory T cells for adoptive immune therapy. These studies will employ novel tools including lentiviral expression systems, directed gene targeting of animals models and human T cells, and the utilization of unique clinical resources to address this question at the site of autoimmune attack in humans with T1D. In sum, data from these Aims are expected to provide information about a key immune checkpoint that impacts autoimmune disease development, and ultimately, lead to novel therapeutic interventions to halt the autoimmune destruction of ? cells in T1D.